Abstact

Glycerol dehydratase (GDHt) enables bioconversion of glycerol to valuable chemicals, but its industrial use is hindered by rapid loss of the adenosylcobalamin (AdoCbl) cofactor (coenzyme B(12)) through both oxygen- and mechanism-based inactivation. To overcome this limitation, we reinforced the AdoCbl-binding interface of Klebsiella pneumoniae GDHt by fusing its alpha and beta subunits with a five-residue linker (fGDHt) and then introducing interface mutations. Fusion alone doubled the oxygen-inactivation half-life without affecting catalytic efficiency. Structural and computational analyses of interface residues, followed by experimental screening, yielded three stabilizing substitutions-alpha-A177M, beta-L113W, and beta-M158W. Pairwise combinations of these mutations yielded double variants whose oxygen-inactivation half-lives increased by up to 24-fold. Enzyme-coupled reactions to convert glycerol into 3-hydroxypropionic acid (3-HP) confirmed that engineered fGDHt variants maintained catalytic activity for longer periods, implying protection against both inactivation modes. In recombinant Escherichia coli strains producing 3-HP, the alpha-A177M/beta-M158W variant matched wild-type titers while operating with 25-fold less AdoCbl. Crystal structures reveal that the mutations tighten inter-subunit packing and, in the case of alpha-A177M, partly occlude an O(2)-access tunnel to the cofactor. These results have established alpha-beta interface engineering as a strategy for engineering more robust GDHts.